Use of il-22 for the treatment of conditions of metabolic disorders

ABSTRACT

The use of IL-22 for the treatment of metabolic disorders including hyperlipidemia, obesity, hyperinsulinemia and diabetics. IL-22 may also be used in combination with insulin for diabetics.

BACKGROUND OF THE INVENTION

Metabolic disorders including obesity, diabetes, hyperlipidemia,hyperglycemia and hyperinsulinemia are complicated syndromes affecting alarge population world wide. It is believed that genetic andenvironmental factors and living habits contributed to the metabolicdisorders. These metabolic disorders contribute significantly to thedevelopment of coronary heart disease. There is no effective treatmentfor metabolic disorders such as for obesity and diabetes. Recombinantinsulin is widely used to effectively control type I diabetes. But fortype II diabetes, treatment with recombinant insulin was not effectivesince patients developed insulin resistance. Insulin sensitizers aredeveloped for type II diabetes. For hyperlipidemia, current treatmentsare mainly focused on reducing cholesterol levels in patients byinhibiting cholesterol synthesis or on inhibiting lipid absorption.

FIELD OF THE INVENTION

The invention is related generally to therapeutics and methods oftreatment. In particular, the present invention relates to therapeuticsand methods of treatment of metabolic disorders, such as obesity,diabetes, hyperlipidemia and hyperinsulinemia.

SUMMARY OF THE INVENTION

It is therefore an object of the present invention to provide analternative therapeutic and method of treatment for medical conditionsassociated with nutritional disorders such as obesity, diabetes andrelated conditions.

The present invention is based on the discovery by the present inventorsthat interleulin-22 (IL-22) modulates a number of conditions oftenrelated to overweight mammals. Accordingly, in one aspect, the inventionprovides a method for the treatment of hyperlipidemia, includingadministering to patients with hyperlipidemia a pharmaceuticallyeffective amounts of IL-22 or a functional derivative thereof. Inanother aspect, the present invention relates to the use of IL-22 or itsfunctional derivative for preparing a medicament for treatinghyperlipidemia.

The invention also provides a method for the reduction of serum glucoselevels in mammals, including giving mammals with hyperglycemia apharmaceutically effective amount of IL-22 or a functional derivativethereof. In another aspect, the present invention relates to the use ofIL-22 or its functional derivative for preparing a medicament fortreating hyperglycemia.

The invention also provides the usage of IL-22 in reducing mammalianserum triglyceride levels and the usage of IL-22 in preparing drugs forreducing mammalian serum triglyceride levels. In another aspect, thepresent invention relates to the use of IL-22 or its functionalderivative for preparing a medicament for modulating serum triglyceridelevels.

In another aspect, the invention provides a method for the treatment ofobesity, including giving obese patients a pharmaceutically effectiveamount of IL-22. In another aspect, the present invention relates to theuse of IL-22 or its functional derivative for preparing a medicament fortreating obesity.

The invention also provides a method for losing weight in mammals,including giving mammals of higher than normal weight a pharmaceuticallyeffective amount of IL-22. The invention also provides the usage ofIL-22 in losing weight in mammals and the usage of IL-22 in preparingdrugs for losing weight in mammals. In another aspect, the presentinvention relates to the use of IL-22 for preparing a medicament forreducing body weight.

Also in another aspect, the invention provides a method for thetreatment of diabetes, including giving patients with diabetes effectiveamount of IL-22. The invention also provides a method for improvingglucose tolerance in mammals, including giving mammals an effectiveamount of IL-22. The invention also provides the usage of IL-22 inimproving mammalian glucose tolerance and the usage of IL-22 inpreparing drugs for improving mammalian glucose utilization. In anotheraspect, the present invention relates to the use of IL-22 or itsfunctional derivative for preparing a medicament for modulating bloodglucose levels.

In a further aspect, the present invention relates to the use of acombination of IL-22 and insulin or their respective derivatives for thetreatment of the above conditions.

The IL-22 in this invention includes but is not limited to human IL-22,recombinant human IL-22, murine IL-22 and/or recombinant murine IL-22.

As used herein, the terminology “consisting essentially of” refers to apolypeptide which includes the amino acid sequence of IL-22 along withadditional amino acids at the carboxyl and/or amino terminal ends whilemaintaining one or more of the biological activities described herein.

Those skilled in the art can readily determine whether a polypeptideconsists essentially of IL-22 under the foregoing definitions bymeasuring the activity of the peptide or polypeptide using the assays asdescribed below.

In the preferred embodiment, the terminology “consisting essentially of”refers to polypeptides which have 8 or less amino acids in addition tothe IL-22 sequence. In the more preferred embodiment, the term means 6or less amino acids in addition to IL-22. In an even more preferredembodiment, the same terminology refers 4 or less amino acids inaddition to IL-22. In another preferred embodiment, the same terminologyrefers to 2 or 1 amino acid in addition to IL-22.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of IL-22 (recombinant mouse IL-22) on totalserum triglyceride levels in normal mice.

FIG. 2 shows the effect of IL-22 on total serum triglyceride levels inacquired obese mice.

FIGS. 3A and 3B show the effect of IL-22 on the adipocyte of fat tissuein acquired (normal aged) obese mice (FIG. 3B) as compared to thecontrol group (FIG. 3A).

FIG. 4 shows the effect of IL-22 on body weight in hereditary (ob/ob)obese mice.

FIG. 5 shows the effect of IL-22 on total serum triglyceride levels inhereditary (ob/ob) obese mice.

FIG. 6 shows the effect of IL-22 on glucose tolerance in mice.

FIG. 7 shows the effect of IL-22 on insulin sensitivity in mice.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The following are examples illustrating various aspects of the presentinvention. All references cited herein are incorporated into thespecification in their entirety.

As used herein, the term “metabolic disorders” is used in a broad senseincluding but not limited to internal imbalances of a human body thatmay be caused by or aggravated by dietary intake of any sort andmultiple environmental and genetic factors. These include medicalconditions such as obesity, diabetes, hyperlipidemia, hyperglycemia andhyperinsulinemia.

Example 1 Human and Murine IL-22 Gene Cloning

The human IL-22 gene cloning used a similar protocol as described in theoriginal cloning paper by Dumoutier et al., PNAS 2000, 97:10144, andincorporated in its entirety herein. Briefly, human peripheral bloodmononuclear cells were cultured for 24 h either with or withoutanti-human CD3 iab (Pharmingen Inc., San Diego). Total RNA was extractedby ultracentrifugation, and cDNA was synthesized with dT being theprimer according to methods described in Molecular Cloning, 2nd edition,(Sambrook et al, Cold Spring Harbor Laboratory Press, 1989). Human IL-22with sequence as shown in SEQ ID NO. 1 was amplified by PCR withspecific primer pair (5′-GCA GAA TCT TCA GAA CAG GTT C-3′ (SEQ ID NO. 5)and 5′-GGC ATC TAA TTG TTA TTT CTA G-3′ (SEQ ID NO. 6)). The amplifiedDNA is cloned to pET21(+) vector and expressed in E. coli strain BL21.

The Mouse IL-22 gene cloning: C57BL/6, female mice were injected withLPS. The spleen was obtained after 20 hours. Total RNA was extracted andcDNA was synthesized through dT primer by the same method describe inhuman IL-22 cloning. Mouse IL-22 was amplified by PCR amplification withspecific primer pair (5′-CTC TCA CTT ATC AAC TGT TGA C-3′ (SEQ ID NO. 7)and 5′-GAT GAT GGA CGT TAG CTT CTC AC-3′ (SEQ ID NO. 8)). The amplifiedcDNA was cloned to pET21(+) vector and expressed in E. coli strain ofBL21.

Both hIL-22 (human IL-22) and mIL-22 (murine IL-22) are certifiedcorrect by DNA sequence. The sequence of murineIL-22 is shown in SEQ IDNO. 2.

Example 2 Recombinant Human IL-22 (RHIL-22) and Recombinant Mouse IL-22(RMIL-22) Protein Expression

The expression of the recombinant proteins used a similar methods asdescribed in the original cloning paper by Dumoutier et al. PNASvol:97:p 10144, 2000 and incorporated herein in its entirety. Briefly E.coli strain BL21(+) (Stratagene) was used as the expression host. Thehost cells were cultured in Luria-Bertani medium with ampicillin (100mg/ml), chloramphenicol (34 mg/ml), and glucose 2%. Expression of theprotein was induced with 1 mM isopropyl-b-D-thiogalactoside. The cellpellet was disrupted with a homogenizer, and the IL-22 inclusion bodieswere obtained by centrifugation. Inclusion bodies were washed withTriszHCl 50 mM, NaCl 100 mM, EDTA 1 mM, DTT 1 mM, and sodiumdeoxycholate 0.5% (wt/vol), pH 8.

Inclusion bodies were solubilized overnight at 4° C. in 8M urea, 50 mM,EDTA 10 mM, and DTT 0.1 mM, pH 6.5. The solution was centrifuged for 1 hat 100,000×g and the supernatant stored at −80° C. until used. Thepurity of the IL-22 was estimated >90% based on SDS-PAGE and Coomassieblue staining analysis. IL-22 protein was refolded by direct dilution ofthe solubilized inclusion bodies in the following folding mixture:

IL-22 100 mg/ml, Tris-HCl 100 mM, EDTA 2 mM, L-arginine 0.5 M, reducedglutathion 1 mM, and oxidized glutathion 0.1 mM, pH 8. The solution wasincubated for 24 hrs at 4° C. The folding mixture was then purifiedusing a Superdex75 (Amersham Pharmacia Biotech) gel filtration column.The protein was eluted with TriszHCl 20 mM and NaCl, 50 mM, pH 7 andstored at −80° C.

Example 3 RMIL-22 Reduced the Total Serum Triglyceride Levels in NormalMice

Normal mice, C57BL6, 8-12 week, female, body weight: 20-25 g (n=10) wereused. The controlled group was injected with a carrier solution (0.1%BSA, Bovine Serum Albumin, PBS, Phosphate Buffered Saline, pH7.0). Thetested group was injected with rm IL-22. The dosages were 0, 3, 30, 100,300 ug/kg/d, once daily, subcutaneous injection continuously for 7 days.Blood samples were collected at different times and the serum wasanalyzed for the determination of total triglyceride levels.

Result: IL-22 can remarkably reduce the total serum triglyceride levelsin normal mice. The effects were dose dependent. FIG. 1 shows theeffects of IL-22 on serum triglyceride levels in normal mice after 7 daytreatment of normal mice with rmIL-22 at 100 ug/kg. The control micewere injected with carrier. The results show that rmL-22 treated grouphad significantly reduced serum triglyceride levels.

Example 4 RMIL-22 Reduced Total Serum Triglyceride Levels andRetroperitoneal Fat Weight in Acquired Obese Mice (Normal Aged Mice)

18-24 month C57BL6 mice, female (n=10), body weight, 30-40 g were used.The controlled group was injected with carrier (0.1% BSA, Bovine SerumAlbumin, PBS, Phosphate Buffered Saline, pH7.0). The tested group wasinjected with rmIL-22 at 300 ug/kg/d, subcutaneously, once daily,continuously for 9 days. Blood was collected at different time and theserum was analyzed for the determination of total triglyceride levels.For the measurement of retroperitoneal fat weight, mice were treated for21 days. Animals were sacrificed at the end of treatment. Results showthat rmIL-22 can remarkably reduce total serum triglyceride levels inacquired obese mice. FIG. 2 shows the effects of IL-22 on serumtriglyceride levels in normal aged mice. Mice were treated with eithercarrier (dashed line) or rmIL-22 (100 ug/kg/day, solid line) for 10days. Mice were 16-month old, C57BL6 mice, female (body weight: 38+/−3gm, n=10). The dotted line indicates controlled group and the solid lineindicates tested group. The results are mean+/−sd.

FIG. 3 shows the histological sections of fat tissues of onerepresentative mouse from each treatment groups. Under the samemagnification, the size of adipocyte in rmIL-22 treated group (FIG. 3B)was significantly smaller than the control treatment group (FIG. 3A). Inaddition, the average weight of fat tissues of tested group (110+/−10mg) is remarkably lower than that of controlled group (175+/−15 mg)(p<0.01).

Example 5 IL-22 Reducing Serum Glucose, Triglyceride, Insulin Levels andBody Weight in Hereditary Obese (OB/OB) Mice

The ob/ob mice, 8-12 week, female, body weight 35-50 g were used. Thecontrolled group was injected with carrier (0.1% BSA, Bovine SerumAlbumin, PBS, Phosphate Buffered Saline, pH7.0). The tested group wasinjected with rmIL-22 300 ug/kg/d subcutaneously, once daily,continuously for 14 days. Body weight (BW) was recorded. The serumlevels for glucose, triglyceride and insulin were determined.

Results showed that rmIL-22 was able to remarkably reduce serum glucose,insulin levels in ob/ob mice. FIG. 4 shows the body weight in ob/ob micetreated with rmIL-22. The dotted line in FIG. 4 indicates controlledgroup and the solid line indicates tested group. In the carrier treatedmice, the body weight increased more than 5% during the 14 daytreatment. In rmIL-22 treated mice, the body weight reduced more than 7%during the 14 day treatment. The data show that rmIL-22 can causereduction of body weight in hereditary obese mice. FIG. 5 shows theeffects of IL-22 on serum triglyceride levels in ob/ob mice. Dashed lineindicates mice treated with carrier and the solid line indicates micetreated with rmIL-22 (300 ug/kg/day) for 2 weeks (n=10). The data showsthat IL-22 can cause reduction of total serum triglyceride levels inhereditary obese mice.

Example 6 rmIL-22 Improving Glucose Tolerance and Insulin Sensitivity inMice

The glucose endurance and insulin sensitivity tests were performed using8-12 week C57BL6 mice, female, body weight: 20-25 g with and withoutrmIL-22 treatment. The controlled group was injected with carrier (0.1%BSA, Bovine Serum Albumin, PBS, Phosphate Buffered Saline, pH7.0). ThermIL-22 treatment group was injected with rmIL-22 300 ug/kg/d, oncedaily, subcutaneous injection continuously for 14 days. At the end oftreatment, mice were fasted over night. Glucose was administered byintraperitoneal injection to each animal at 2 mg/g (body weight). Bloodglucose concentration was determined at 30, 60 and 120 mins post Glucoseinjection. FIG. 6 shows that IL-22 enhanced the glucose utilization inmice in a glucose tolerance test. Dashed line indicates blood glucoselevels in mice treated with carrier. Solid line indicates glucose levelsin mice treated with rmIL-22 (300 ug/kg/day) for 2 weeks (n=10). Glucoselevels are significantly different between the two groups at 60 minutesand 120 minutes (p<0.01).

FIG. 7 shows that rmIL-22 can increase the insulin sensitivity in mice.The dotted line indicates controlled group and the solid line indicatesrmIL-22 (300 ug/kg/day) treated group for 2 weeks. Both groups ofanimals were injected with the same dosage of insulin, and the bloodglucose levels were measured after insulin treatment. Glucose levels,shown as percentage change in nnIL-22 treated group are significantlylower than the control treatment group at 60 minutes and 120 minutes(p<0.01).

While the various aspects of the present invention have been illustratedusing the examples described above, it is clear that there are manyother ways that the present invention may be practiced given theguidance provided herein. For example, the hosts that can be used toexpress or clone IL-22 in this invention include prokaryotic cells,yeast cells or eukaryotic cells. The proper prokaryotic cells includebut are not limited to G+/G− bacteria, such as E. coli. These areavailable E. coli strains: K12 MM294 (ATCC 31,446), X1776 (ATCC 31,537),W3110 (ATCC 27,325) and K5 772 (ATCC 53,635), JM109, DH5, B stains,B834, BL21, BLR et al. Other proper prokaryotic cells include: Erwinia,Klebsiella, Proteus, Salmonella, such as Salmonella typhimurium,Serratia, Shigella, B. subilis, B. licheniformis, Pseudoinonas,Streptomyces. Among numbers of available E. coli strains, E. coli. W3110is the first to be chosen for recombinant DNA products.

Besides prokaryotic cells, eukaryotic cells as filamentous fungi oryeast cells are also proper for the expression or clone of IL-22 in thisinvention. These are proper strains: Saccharomyces, Schizosaccharomycespombe (Beach and Nurse, Nature, 290:140-142 (1981); EP 139,383),Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Flee et al.,Bio/Technology, 9:968-975 (1991), such as K lactis (MW 98-8C, CBS683,CBS4574; Louvencourt et al., J. Bacteriol., 154(2):737-742 (1983)), K.fragilis (ATCC 12,424), K. waltii (ATCC 56,500), K. drosophilarum (ATCC36,906; Van den Berg et al., BioTechnology, 8:135-139 (1990)), K.thermotolerans, K. marxianus; yarrowia (EP 244,234), Neurospora crassa(Case et al., Proc. Natl. Acad. Sci. USA, 76:5259-5263 (1979)),schwanniomyces as Schwanniomyces occidentalis (EP 394,538), filamentousfungi as Neurospora, penicilliun, Tolypocladium (WO 91/00357),Aspergillus as A. nidulans (Balance et al., Biochem. Biophys. Res.Commum., 112:284-289 (1983); Tilburn et al., Gene, 26:205-221 (1983);Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474 (1984)), A.niger (Kelly and Hynes, EMBO J., 4:475-479 (1985)). Methylotropic yeastsare also proper, including those ones that can grow on methanol, such asHansenula, Candida, kloeckera, Pichia, Saccharomyces, Torulopsis,Rhodotorula. (C. Anthony, The Biochemistry of Methylotrophs, 269(1982)).

The host used to express glycosylated IL-22 in this invention arepreferably from multicell organisms. The Invertebrate cells includeinsect cells as Drosophila 82 and Spodoptera Sf9 and plant cells. Theproper mammalian cells include CHO, COS cells, especially CV1 straintransformed by SV40 (COS-7, ATCC CRL 1651), human embryo renal cells 293(Graham et al., J. Gen. Virol., 36:59-74 (1977)), CHO/-DHFR (Urlaub andChasin, Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980)), murine testestrophoblast cells (TM4, Mather, Biol. Reprod., 23:243-251 (1980)), humanpulmonary cells (WI38, ATCC CCL 75), human liver cells (hep G2, KB8065), murine mastocarcinoma cells (MMT 060562, ATCC CCL 51).Technicians in this field should know how to select proper host cells.

After transfection or conversion by the vector, the above host cells canbe cultured in common nutrient media. The modified media are fit forinducing the promoter, selecting transformant or amplifying IL-22encoded gene sequence. Technicians in this field should know the cultureconditions, such as media, temperature, pH, et al. Detailed techniquescan be seen in Mammalian Cell Biotechnology: A Practical Approach, M.Butler, ed. (IRL Press, 1991) and Sambrook et al., supra.

The method of eukaryotic transfection and prokaryotic conversion, suchas CaCl2 method, calcium phosphate precipitation, liposome intermediaryor electroporation are known to those skilled in the art. For example,CaCl2 method (Sambrook et al., supra.) or electroporation are fit forprokaryotic cells. The infection of Agrobacterium tu iefaciens is fitfor some plant cells (Shaw et al., Gene, 23:315-330 (1983) and WO89/05859). Calcium phosphate precipitation is fit for mammalian cells(Graham and van der Eb, Virology, 52; 456-457 (1978)). Detailedtechniques on yeast cell conversion can be seen in Van Solingen et al.,J. Bact., 130:946-947 (1977); and Hsiao et al., Proc. Natl. Acad. Sci.(USA), 76:3829-3833 (1979). Other method such as microinjection ofnucleic acid, electroporation, bacterial protoplast fusion with intactcells et al are also fitful. Techniques concerned with conversion ofmammalian cells can be seen in Keown et al., Methods in Enzymology,185:527-537 (1990); and Mansour et al., Nature, 336:348-352 (1988).

The DNA sequence encoding IL-22 in this invention can be inserted into areplicable vector to clone the gene or express the protein. All thevectors, such as plasmid, cosmid, virion or bacteriophage are publiclyavailable. Applying common techniques in this field, technicians caninsert the DNA sequence encoding IL-22 into appropriate restrictionendonuclease sites. A replicable vector usually contains but is notlimited to following parts: one or more signal sequence, one origin ofreplication, one or more marker gene, one enhancer element, onepromoter, and one transcription termination sequence. Applying standardligation techniques in this field, technicians can construct anappropriate replicable vector containing one or more above parts.

The IL-22 in this invention can be directly expressed throughrecombinant DNA, and it can also be produced through fusion ofpolypeptides. The later can be a signal sequence localized in the matureprotein or N-terminal of the polypeptide. It can also be other fragmentswith special cutting sites localized in the mature protein or N-terminalof the polypeptide. Usually, the signal sequence is one part of theabove replicable vector, or one part of DNA sequence encoding IL-22 inthis invention. The signal sequence can be prokaryotic one, such as ALP,penicillinase, lpp, etc. In yeast secretion, the signal sequence can beyeast invertase leader, a agent leader sequence or ALP leader sequence,etc. In mammalian expression, the mammalian signal sequence can bedirectly used to secrete the target protein.

Both the expression vector and the clone vector have a piece of DNAsequence, which can make the vector replicate in one or more hosts. Thesequence corresponding with bacteria, yeast and virus hosts are known totechnicians in this field (Molecular Cloning, 2nd edition, (Sambrook etal, Cold Spring Harbor Laboratory Press, 1989).

Both the expression vector and the clone vector have a piece ofselecting gene, that is “selecting marker”. Typical protein expressed byselecting gene has resistance to some antibiotics and toxin, is able toremedy auxotrophic deficiencies and supplement some key nutrient factorsthat complex media can not provide.

The selecting gene suitable for mammalian host cells may be able todistinguish the host cells containing IL-22 encoding gene, such as DHFRor thymidine kinase. The proper host cell with wide DHFR as selectinggene is CHO strain without DHFR activity. The culture method can be seenin Urlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216-4220 (1980). Theselecting gene suitable for yeast cells is trp1 gene expressed in yeastplasmid Yrp7 (Stinchcomb et al., Nature, 282:39-43 (1979); Kingsman etal., Gene, 7:141-152 (1979); Tschumper et al., Gene, 10:157-166 (1980)).Trpl gene can be used to select yeast mutation strain which can not growon tryptophan, such as ATCC No. 44047 or PEP4-1 (Jones, Genetics,85:22-33 (1977)).

Both expression vector and clone vector usually have a promoter that canbe ligated to the IL-22 encoding DNA sequence, which can direct mRNAsynthesis. Promoters corresponding with all kinds of hosts should beknown to technicians in this field. The promoters suitable forprokaryotic hosts include beta-lactamase and lactose promoter system(Chang et al., Nature, 275:615 (1978); Goeddel et al., Nature, 281;544-548 (1979)), ALP and trp promoter system (Goeddel, Nucleic AcidsRes., 8:4057-4074 (1980); EP 36,776), hetero-promoter as tac promoter(deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)). Bacterialpromoter also has a piece of Shine-Dalgamo sequence that can be ligatedto the IL-22 encoding sequence.

Promoters suitable for yeast host include 3-phosphoglycericphosphokinase promoter (Hitzeman et al., J. Biol. Chem., 255:12073-12080(1980)) or other glycolysis enzyme promoters (Hess et al., J. Adv.Enzyme Reg., 7:149-167 (1968); Holland, Biochemistry, 17:4900-4907(1978)), such as enolase, glyceraldehydes-3-phosphatedehydrogenase,hexokinase, pyruvate decarboxylase, fructose diphosphatase, et al.

Some other inducible yeast promoter can regulate transcription accordingto different growing conditions. Detailed description can be seen inU.S. Pat. Nos. 5,063,154 and 6,221,630.

Promoters can control the transcription of IL-22 encoding gene on thereplicable vector in mammalian host cells. The promoters include onesfrom certain viral genome, foreign mammalian promoters and heat shockprotein promoter, et al. However, those promoters should be compatiblewith the expression system of the host.

The transcription of the IL-22 encoding sequence in eukaryoticexpression system can be enhanced through the insertion of enhancer intothe replicable vectors. Enhancer is a kind of cis-acting element of DNAmolecules, usually 10-300 bp, which can enhance the transcription of DNAmolecules by acting on the promoters. Numbers of enhancers have beenknown from mammalian gene, and most widely used enhancers are fromeukaryotic viral cells. The enhancers can be inserted into 5′ or 3′terminal of the IL-22 encoding sequence on the replicable vectors but 5′terminal is first chosen.

The expression vectors in eukaryotic host cells (yeasts, fungi, insects,plants, animals, human, or other multicell organisms) also contain theDNA sequence for terminating transcription and stabilizing mRNA. Thiskind of sequence is usually from the 5′ or 3′ terminal of non-translatedregion in eukaryotic cells or viral DNA or cDNA. Other methods, vectorsand hosts can be seen in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; EP 117,058.

Gene marking and gene therapy may be performed using the protocolstaught by Anderson et al., Science 256, 808-813 (1992). The genedelivery system may include various viral-based vectors includingretroviral vectors, adenoviral vectors or AAV, director plasmiddelivery, of liposome-mediated or receptor-mediated gene delivery (Dzauet al., Trends in Biotechnology 11, 205-210, 1993).

The IL-22 encoding DNA sequence in this invention can be used on genetherapy, in the course of which the IL-22 gene is transduced into cellsin order to express the product having therapeutic effects, such asreplacing the former defective gene. Gene therapy includes traditionaltherapy, that is long term effects through one time therapy and givinggene therapy drugs. The later includes giving effective DNA or mRNA onetime or several times.

The IL-22 in this invention can be used as drugs. Technicians in thisfield can prepare several kinds of effective formulation according tousual method, which contains effective amount of IL-22 and medicinalcarriers.

When prepared as lyophilization or liquid, the medicinal composition inthis invention should be added some other carrier, excipient,stabilizer, et al that are acceptable physiologically for theconvenience of preservation such as described in Remington'sPharmaceutical Sciences 16th edition, Osol, A., Ed. (1980)), thedisclosure of which is incorporated by reference herein in its entirety.The dosage and concentration of the carrier, excipient and stabilizershould be safe for human, mice and other mammals.

In one aspect, the formulation comprises an effective amount of IL-22and a physiologically acceptable carrier, diluent, or excipient, or acombination thereof.

The term “pharmaceutical composition” or “pharmaceutical formulation”refers to a mixture of IL-22 with other chemical components, such asdiluents or carriers. The pharmaceutical composition facilitatesadministration of the IL-22 to an organism. Multiple techniques ofadministering the formulation may be used including, but not limited to,oral, injection, aerosol, parenteral, and topical administration.

The term “carrier” defines a chemical compound that facilitates theincorporation of the IL-22 into cells or tissues. For example dimethylsulfoxide (DMSO) is a commonly utilized carrier.

The term “diluent” defines chemical compounds diluted in water that willdissolve the IL-22 as well as stabilize the biologically active form ofthe IL-22. Salts dissolved in buffered solutions are utilized asdiluents in the art. One commonly used buffered solution is phosphatebuffered saline because it mimics the salt conditions of human blood.Since buffer salts can control the pH of a solution at lowconcentrations, a buffered diluent rarely modifies the biologicalactivity of a pharmaceutically active substance.

The term “physiologically acceptable” defines a carrier or diluent thatdoes not abrogate the biological activity and properties of the IL-22.

The pharmaceutical compositions described herein can be administered toa human patient per se, or in pharmaceutical compositions where they aremixed with other active ingredients, as in combination therapy, orsuitable carriers or excipient(s). Techniques for formulation andadministration of the formulations described in the instant applicationmay be found in “Remington's Pharmaceutical Sciences,” Mack PublishingCo., Easton, Pa., 18th edition, 1990, the disclosure of which isincorporated herein by reference in its entirety.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, or intestinal administration; parenteral delivery,including intramuscular, subcutaneous, intravenous, intramedullaryinjections, as well as intrathecal, direct intraventricular,intraperitoneal, intranasal, or intraocular injections.

Alternately, one may administer the IL-22 formulation in a local ratherthan systemic manner, for example, via injection directly in the renalor cardiac area, often in a depot or sustained release formulation.Furthermore, one may administer the IL-22 in a targeted drug deliverysystem, for example, in a liposome coated with a tissue-specificantibody. The liposomes will be targeted to and taken up selectively bythe organ.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or tabletting processes.

Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of IL-22 into preparations whichcan be used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen. Any of the well-known techniques,carriers, and excipients may be used as suitable and as understood inthe art; e.g., in Remington's Pharmaceutical Sciences, above.

For injection, the IL-22 of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks's solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the IL-22 can be formulated readily bycombining it with pharmaceutically acceptable carriers well known in theart. Such carriers enable the IL-22 to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Pharmaceuticalformulations for oral use can be obtained by mixing one or more solidexcipient with IL-22, optionally grinding the resulting mixture, andprocessing the mixture of granules, after adding suitable auxiliaries,if desired, to obtain tablets or dragee cores. Suitable excipients are,in particular, fillers such as sugars, including lactose, sucrose,mannitol, or sorbitol; cellulose preparations such as, for example,maize starch, wheat starch, rice starch, potato starch, gelatin, gumtragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodiumcarboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired,disintegrating agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodiumalginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of IL-22 doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the IL-22 in admixture with filler such as lactose,binders such as starches, and/or lubricants such as talc or magnesiumstearate and, optionally, stabilizers. In soft capsules, the IL-22 maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid paraffin, or liquid polyethylene glycols. In addition,stabilizers may be added. All formulations for oral administrationshould be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the IL-22 for use according to thepresent invention is conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the IL-22 and asuitable powder base such as lactose or starch.

IL-22 may be formulated for parenteral administration by injection,e.g., by bolus injection or continuous infusion. Formulations forinjection may be presented in unit dosage form, e.g., in ampoules or inmulti-dose containers, with an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the IL-22 in water-soluble form. Additionally, insome embodiments, suspensions of the IL-22 may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of theIL-22 to allow for the preparation of highly concentrated solutions.

Alternatively, the IL-22 may be in powder form for constitution with asuitable vehicle, e.g., sterile pyrogen-free water, before use.

The IL-22 may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the IL-22 may alsobe formulated as a depot preparation. Such long acting formulations maybe administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theIL-22 may be formulated with suitable polymeric or hydrophobic materials(for example as an emulsion in an acceptable oil) or ion exchangeresins, or as sparingly soluble derivatives, for example, as a sparinglysoluble salt.

If desired, the formulation may comprise a cosolvent system comprisingbenzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer,and an aqueous phase. A common cosolvent system used is the VPDco-solvent system, which is a solution of 3% w/v benzyl alcohol, 8% w/vof the nonpolar surfactant Polysorbate 80™, and 65% w/v polyethyleneglycol 300, made up to volume in absolute ethanol. Naturally, theproportions of a co-solvent system may be varied considerably withoutdestroying its solubility and toxicity characteristics. Furthermore, theidentity of the co-solvent components may be varied: for example, otherlow-toxicity nonpolar surfactants may be used instead of POLYSORBATE80™; the fraction size of polyethylene glycol may be varied; otherbiocompatible polymers may replace polyethylene glycol, e.g., polyvinylpyrrolidone; and other sugars or polysaccharides may substitute fordextrose.

Alternatively, if desired other delivery systems may be used. Liposomesand emulsions are well known examples of delivery vehicles or carrierswhich may be utilized. Certain organic solvents such asdimethylsulfoxide also may be employed, although usually at the cost ofgreater toxicity. In some embodiments, the IL-22 may be delivered usinga sustained-release system, such as semipermeable matrices of solidhydrophobic polymers containing the therapeutic agent. Varioussustained-release materials have been established and are well known bythose skilled in the art. Sustained-release capsules may, depending ontheir chemical nature, release the IL-22 for a few weeks up to over 100days. Depending on the chemical nature and the biological stability ofthe therapeutic reagent, additional strategies for protein stabilizationmay be employed.

In some embodiments, the IL-22 may be provided as a salt withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but not limited tohydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free acid or base forms.

Pharmaceutical compositions suitable for use in the present inventioninclude compositions where the IL-22 is contained in an amount effectiveto achieve its intended purpose. More specifically, a therapeuticallyeffective amount means an amount of IL-22 effective to prevent,alleviate or ameliorate symptoms of disease or prolong the survival ofthe subject being treated. Determination of a therapeutically effectiveamount is well within the capability of those skilled in the art.

The exact formulation, route of administration and dosage for the IL-22compositions of the present invention can be chosen by the individualphysician in view of the patient's condition. (See e.g., Fingl et al.1975, in “The Pharmacological Basis of Therapeutics”, Ch. 1 p. 1). Thedosage may be a single one or a series of two or more given in thecourse of one or more days, as is needed by the patient. In someembodiments, a suitable human dosage can be inferred from ED₅₀ or ID₅₀values, or other appropriate values derived from in vitro or in vivostudies, as qualified by toxicity studies and efficacy studies inanimals.

Dosage amount and interval may be adjusted individually to provideplasma levels of the Il-22 which are sufficient to maintain themodulating effects, or minimal effective concentration (MEC). Ifdesired, the MEC can be estimated from in vitro data. Dosages necessaryto achieve the MEC will depend on individual characteristics and routeof administration. However, HPLC assays or bioassays can be used todetermine plasma concentrations.

Dosage intervals can also be determined using MEC value. In someembodiments, the formulations are administered using a regimen whichmaintains plasma levels above the MEC for 10-90% of the time, preferablybetween 30-90% and most preferably between 50-90%.

In cases of local administration or selective uptake, the effectivelocal concentration of the IL-22 may not be related to plasmaconcentration.

The amount of composition administered will, of course, be dependent onthe subject being treated, on the subject's weight, the severity of theaffliction, the manner of administration and the judgment of theprescribing physician.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration. The pack or dispensermay also be accompanied with a notice associated with the container inform prescribed by a governmental agency regulating the manufacture,use, or sale of pharmaceuticals, which notice is reflective of approvalby the agency of the form of the drug for human or veterinaryadministration. Such notice, for example, may be the labeling approvedby the U.S. Food and Drug Administration for prescription drugs, or theapproved product insert. Compositions according to the present inventionformulated in a compatible pharmaceutical carrier may also be prepared,placed in an appropriate container, and labeled for treatment of anindicated condition.

In some embodiments, the composition may include buffer, antioxidant,small polypeptide, proteins such as serum albumin, gelatin orimmunoglobulin, hydrophilic polymers such as PVP, amino acids, such asaminoacetate glutamate salt, etc., glycose, biose and other carbohydrateas glucose, mannose or dextrin, chelate agent as EDTA, sugar alcohols asmannitol, sorbitol, counterions as Na+, and/or nonionic surfactants suchas TWEEN™, PLURONICS™ or PEG, etc.

Preferably, the preparation containing IL-22 in this invention issterilized before injection. This procedure can be done using sterilefiltration membranes before or after lyophilization and reconstitution.

The medicinal composition is usually filled in a container with sterileaccess port. The medicinal composition in this invention can be injectedthrough normal ways, including but not limited to intravenous,intra-abdominal, intracephalic, intramuscular, intraocular,intra-arterial, locally or through sustained release systems.

The dosage and concentration can be adjusted according to actualsituation. Technicians in this field should know how to choose properdosage and injection ways according to actual situation. The animaltests in this invention have provided believable direction for theeffective amount in human body. The adjustment principle of betweendifferent species can be seen in Mordenti, J. and Chappell, W. “The useof interspecies scaling in toxicokinetics” In Toxicokinetics and NewDrug Development, Yacobi et al.; Pergamon Press, New York 1989, pp.42-96.

When the IL-22 is injected in mammals, the usual dosage is 1 ng/kg-100mg/kg body weight daily, optimally 10 ug/kg/d-100 ug/kg/d. The dosageshould be adjusted according to different injection way. The directioncan be seen in U.S. Pat. Nos. 4,657,760; 5,206,344; or 5,225,212.Predictably, different IL-22 preparations effect on different diseases.When the effecting target (organ or tissue) changes, the injection wayshould be adjusted accordingly.

The micro-capsule containing IL-22 can be used as sustained releasesystem. Techiques of micro-capsule sustained release system ofrecombinant protein have been successfully adopted on rhGH, rhIFN, IL-2and MNrgp120 (Johnson et al., Nat. Med., 2:795-799 (1996); Yasuda,Biomed. Ther 27:1221-1223 (1993); WO 97/03692, WO 96/40072, WO 96/07399;U.S. Pat. No. 5,654,010).

The sustained release system of IL-22 in this invention can be preparedwith PLGA which is biologically compatible and easily degraded. Lactateand hydroxyacetate, the degrading products of PLGA, can be clearedquickly in human body. Furthermore, the degrading ability can bedifferent from several months to several years according to itsdifferent molecule and composition (Lewis, “Controlled release ofbioactive agents form lactide/glycolide polymer,” in: M. Chasin and R.Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (MarcelDekker: New York, 1990), pp. 141)).

The IL-22 in this invention can be modified with activated PEG with, forexample, but not limited to, molecular weight being 5,000-100,000 forthe purpose of prolonging its half-life time. It can also be prepared asChimeric Molecule or Fusion Protein, for the purpose of enhancing itsbiological activity or prolonging its half-life time. Detailedtechniques can be seen in Greenwald et al., Bioorg. Med. Chem. Lett.1994, 4:2465-2470; Caliceti et al., IL Farmaco, 1993, 48:919-932, etc.

IL-22 in this invention can be prepared as a chimeric molecule or fusionprotein for the purpose of enhancing its biological activity orprolonging its biological half-life time. Covalently modified IL-22 isalso included in this invention. Chemically covalent modificationincludes modifying N or C terminal or adding a chemical molecule to theother amino acid. It also includes modification of amino acid sequence,modification of the glycosylation of IL-22 itself.

Other preparing techniques as nanotechnology preparation (U.S.60/544,693), spraying preparation (for example as taught inCN00114318.2, PCT/CN02/00342), inhaling preparation, et al. are alsoincluded in this invention. All cited references are hereby incorporatedin their entirety.

The polypeptide used for the above experiments contains the sequence asshown in SEQ ID NO. 1 using the nucleic acid sequence as shown in SEQ IDNO. 3. Nevertheless, it is clear that SEQ ID NOs. 1-4 are only someembodiments of the present invention and that the same principle of thepresent invention can also be applied to other functionally equivalentpeptides that have been modified without affecting the biologicalfunction of IL-22. For example, those with conservative amino acidsubstitutions (i.e. amino acids within the same biochemical type such ashydrophobic, hydrophilic, positive or negatively charged groups). Thosepeptides that have one or more of the above modification and yet retainthe activity described in the present invention are referred to asfunctional variants. Other peptides that have at least 95%, 90%, 85%,80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35% or 30% amino acid ornucleotide sequence identity to one of SEQ ID NOs. 1-4 and retain one ormore activities of IL-22 or encode a polypeptide which retains one ormore activities of IL-22 are also considered variants.

Amino acid or nucleotide sequence identity may be evaluated using any ofthe variety of sequence comparison algorithms and programs known in theart. Such algorithms and programs include, but are by no means limitedto, TBLASTN, BLASTP, FASTA, TFASTA, and CLUSTALW (Pearson and Lipman,1988, Proc. Natl. Acad. Sci. USA 85(8):2444-2448; Altschul et al., 1990,J. Mol. Biol. 215(3):403-410; Thompson et al., 1994, Nucleic Acids Res.22(2):4673-4680; Higgins et al., 1996, Methods Enzymol. 266:383-402;Altschul et al., 1990, J. Mol. Biol. 215(3):403-410; Altschul et al.,1993, Nature Genetics 3:266-272). In a particularly preferredembodiment, protein and nucleic acid sequence homologies are evaluatedusing the Basic Local Alignment Search Tool (“BLAST”) which is wellknown in the art (see, e.g., Karlin and Altschul, 1990, Proc. Natl.Acad. Sci. USA 87:2267-2268; Altschul et al., 1990, J. Mol. Biol.215:403-410; Altschul et al., 1993, Nature Genetics 3:266-272; Altschulet al., 1997, Nuc. Acids Res. 25:3389-3402). The BLAST programs identifyhomologous sequences by identifying similar segments, which are referredto herein as “high-scoring segment pairs,” between a query amino ornucleic acid sequence and a test sequence which is preferably obtainedfrom a protein or nucleic acid sequence database. High-scoring segmentpairs are preferably identified (i.e., aligned) by means of a scoringmatrix, many of which are known in the art. Preferably, the scoringmatrix used is the BLOSUM62 matrix (Gonnet et al., 1992, Science256:1443-1445; Henikoff and Henikoff, 1993, Proteins 17:49-61). Lesspreferably, the PAM or PAM250 matrices may also be used (see, e.g.,Schwartz and Dayhoff, eds., 1978, Matrices for Detecting DistanceRelationships Atlas of Protein Sequence and Structure, Washington:National Biomedical Research Foundation). The BLAST programs evaluatethe statistical significance of all high-scoring segment pairsidentified, and preferably selects those segments which satisfy auser-specified threshold of significance, such as a user-specifiedpercent homology. Preferably, the statistical significance of ahigh-scoring segment pair is evaluated using the statisticalsignificance formula of Karlin (see, e.g., Karlin and Altschul, 1990,Proc. Natl. Acad. Sci. USA 87:2267-2268).

The BLAST programs may be used with the default parameters or withmodified parameters provided by the user.

Other variants include modifications such as conjugation of othermaterial to IL-22 some examples of which are described above and arealso considered functional derivatives of IL-22.

It will be understood by those of skill in the art that numerous andvarious modifications can be made without departing from the spirit ofthe present invention. Therefore, it should be clearly understood thatthe forms of the present invention are illustrative only and are notintended to limit the scope of the present invention.

1. A method of treating a metabolic disorder, or a medical condition ordisease associated therewith, comprising administering to a subject inneed thereof a therapeutically effective amount of a polypeptideconsisting essentially of IL-22 or a known functional derivativethereof.
 2. The method according to claim 1, wherein the metabolicdisorder or medical condition or disease comprises obesity, diabetes,hyperlipidemia, hyperglycemia or hyperinsulinemia.
 3. The methodaccording to claim 1, further comprising administering a therapeuticallyeffective amount of insulin or a known functional derivative thereof. 4.The method according to claim 1, wherein the polypeptide consistingessentially of IL-22 is provided with, and expressed by, apharmaceutically acceptable and therapeutically effective expressionsystem.
 5. The method according to claim 4, wherein the expressionsystem synthesizes an in vivo effective amount of the polypeptideconsisting essentially of IL-22.
 6. The method according to claim 1,wherein the polypeptide consists essentially of human IL-22 or mouseIL-22.
 7. A method of reducing body weight in a subject, said methodcomprising administering to sadi subject an effective amount of IL-22.8. Use of IL-22 or functional derivatives thereof for the manufacture ofa medicament for the treatment of metabolic disorders.
 9. Use of IL-22for the manufacture of a medicament for the treatment of maetabolicdisorders.
 10. The use according to claim 9 wherein said metabolicdisorder comprises a condition selected form a group consisting ofobesity. diabetes, hyperlipidemia, hyperglycemia and hyperinsulinemia.11. Use of IL-22 or functional derivatives thereof for the manufactureof a medicament for the reduction of body weight.
 12. Use of IL-22 forthe manufacture of a medicament for the reduction of body weight. 13.Use a combination of IL-22 and insulin for the treatment of bloodglucose disorders.
 14. A method of treating a metabolic disorder, ormedical condition or disease associated therewith, comprising deliveringto a subject in need of treatment a polynucleotide comprising a DNA thatencodes a polypeptide consisting essentially of IL-22 delivery system;providing an expression system for the polypeptide encoding DNA; andallowing the in vivo expression of a therapeutic amount of thepolypeptide.
 15. The method of claim 14, wherein the metabolic disorderor medical condition or disease comprises or is associated with obesity,diabetes, hyperlipidemia, hyperglycemia or hyperinsulinemia.
 16. Themethod of claim 14, wherein the gene delivery system comprises at leastone of a retroviral vector(s), adenoviral vector(s), orliposome-mediated director plasmid delivery, or receptor-mediated genedelivery.
 17. The method of claim 14, which comprises gene therapyalone, or in combination with another known therapy(ies).
 18. The methodof claim 14, further comprising administering a therapeuticallyeffective amount of insulin or a known functional derivative thereof.19. The method of claim 14, wherein the expression system synthesizes anin vivo effective amount of the polypeptide consisting essentially ofIL-22.
 20. The method of claim 14, wherein the polypeptide consistsessentially of human IL-22 or mouse IL-22.